Genes homeobox, vitais para muitos aspectos relacionados com crescimento e diferenciação celular, têm sido descritos desregulados em alguns cânceres. Seu papel na carcinogênese, principalmente de carcinomas epidermóides de boca, permanence pouco claro e pobremente caracterizado. Desse modo, esse estudo objetivou avaliar, em cultura de células, o perfil de expressão de seis genes homeobox (ASH2L, HOXA7, HHEX, PKNOX1, PITX1, TGIF) selecionados dentre aqueles previamente identificados no Projeto Genoma Câncer de Cabeça e Pescoço (2001) sob estímulo de EGF e TGF-beta1. Para tal, linhagens celulares de carcinoma epidermóide de cabeça e pescoço primário (HN6) e metastático (HN31) e uma linhagem não-tumoral (HaCat) foram cultivadas sob condições-padrão. Após a confecção dos cDNAs de cada linhagem, por meio de RT-PCR, os transcritos foram amplificados e quantificados pela técnica de PCR em tempo real. Os dados foram normalizados com o gene HPRT e a quantificação relativa foi realizada seguindo o método do delta Ct. De acordo com os resultados foi possível verificar que o EGF produziu uma modulação variável da expressão dos genes avaliados em todas as linhagens celulares, enquanto que, em geral, o TGF-beta1 foi capaz de aumentar significantemente (ANOVA, p<0,05) a expressão dos transcritos de 5 genes homeobox (HOXA7, HHEX, PKNOX1, PITX1, TGIF). Particularmente transcritos dos genes PITX1 e TGIF foram signicantemente mais expressos nas linhagens tumorais (HN6 e HN31) frente à linhagem não-tumoral quando tratados com TGF-beta1. Desse modo, sugere-se que os genes homeobox estudados desempenhem diferentes funções na carcinoma epidermóide de boca, e que, especialmente PITX1 e TGIF atuem como oncogenes inibindo a resposta anti-proliferativa dependente de TGF-beta e levando a progressão tumoral. / Homeobox genes, vital to many aspects related with cellular growth and differentiation, had been described as deregulated in some cancers. Their role in carcinogenesis, mainly oral squamous cell carcinomas, remains unclear and poorly characterized. Thus, this study had the purpose to evaluate, in cell cultures, the expression profile of six homeobox genes (ASH2L, HOXA7, HHEX, PKNOX1, PITX1, TGIF) selected among genes previously identified in the Head and Neck Cancer Genoma Project (2001), under stimulation with EGF and TGF-beta1. Oral squamous cell carcinoma cell lines from primary tumour (HN6) and from methastasis (HN31), and a non-tumoral cell line (HaCat) were cultured under standard procedures. CDNAs were obtained by RT-PCR and the transcripts were amplified and quantified by real-time PCR. Data were normalized by HPRT gene and the relative quantification was made by the delta Ct method. According to the results, it was possible to observe that EGF produced a variable modulation of the analyzed genes, in all cell lines. Generally, TGF-beta1 was able to significantly increase (ANOVA, p<0,05) the expression of the transcripts of 5 homeobox genes (HOXA7, HHEX, PKNOX1, PITX1, TGIF). Transcripts of PITX1 and TGIF genes were particularly more expressed in the tumoral cell lines (HN6 e HN31), when compared to the non-tumoral cell line, when treated with TGF-beta1. It is suggested that the studied homeobox genes play different roles in oral squamous cell carcinoma and that, especially the PITX1 and TGIF act as oncogenes, inhibitting the TGF-dependent anti-proliferative response, leading to tumour progression.

Como neoplasia maligna que mais acomete a cavidade oral, o carcinoma epidermóide gera infinidade de estudos acerca de sua gênese e progressão. O variado perfil genético e protéico observado leva a freqüente insucesso nas terapias adotadas, contribuindo para pobres prognósticos. Este estudo visa compreender melhor o papel da proteína Akt, tida como chave para a proliferação de diversas neoplasias, frente ao estímulo das células derivadas de carcinoma epidermóide humano em cultura (HaCat, HN6, HN19, HN30, HN31) com EGF 10ng/ml e TGF &#946; 5ng/ml. Para tanto, analisou-se a expressão de pAkt, ciclina D1, Bad, caspase-3 e PTEN, através de imunofluorescência, western-blot e imunoprecipitação. Os resultados mostraram aumento da expressão de pAkt e ciclina D1 frente ao tratamento com EGF, bem como diminuição de Bad e PTEN, com exceção de HN31. A imunoprecipitação revelou neste caso que pAkt previne a apoptose através de inativação direta de Bad. Já nas células tratadas com TGF &#946; , obtivemos resultados diferentes do esperado para este supressor de tumor. A expressão de pAkt mostrou-se aumentada nas linhagens celulares, mas estável em HN19 e HN31. Ciclina D1 mostrou aumento em HN6 e HN30, manutenção em HaCat e HN19 e diminuição em HN31. HaCat mostrou queda dos níveis de caspase-3 e Bad, manutenção em HN6, aumento em HN30, aumento somente de Bad em HN31 e HN19 com níveis inalterados após o tratamento para ambas as proteínas. Estes achados sugerem o importante papel desempenhado pelo EGF nas linhagens de carcinoma epidermóide estudadas, e como esta via é importante candidata a ser visada em tratamentos quimioterápicos. A ação do TGF &#946; mostrou discrepâncias, revelando seu comportamento dúbio frente os diversos tipos celulares, e sugerindo possível relação entre este receptor e a via do pAkt, o que requer estudos mais apropriados. A baixa ocorrência de apoptose também reforça esta possibilidade, mostrando como a via do Akt é essencial para a progressão neoplásica e pode estar relacionada a mais eventos celulares do que já sabido. / Squamous cell carcinoma raises a great interest regarding carcinogenesis and its proliferative pathways, due to the high incidence and poor prognosis. The broad genetic and proteic profiles contribute to this poor prognosis. The present study aims to better comprehend the role played by pAkt, key protein for the development of many neoplasms. Cell lines derived from oral squamous cell carcinoma (HaCat, HN6, HN19, HN30 and HN31) were induced with 10ng/ml EGF and 5ng/ml TGF &#946; . The expressions of pAkt, cyclin D1, Bad, caspase-3 and PTEN were analyzed through immunofluorescence, western-blot and immunoprecipitation. Results showed higher pAkt and cyclin D1 expression after EGF treatment, as well as a decrease in Bad and PTEN levels, except for HN31. Immunoprecipitation revealed that pAkt prevents apoptosis through Bad direct inactivation. However, TGF &#946; treatment revealed different results, from what expected for this tumor supressor. pAkt expression revealed to be increased in all cells lines, but stable for HN19 and HN31. HaCat exhibited decreased levels of caspase-3 and Bad, which were unaltered in HN6, augmented in HN30. HN31 revealed only Bad increased levels, and no alterations in HN19. These findings suggest the crucial role played by EGF stimulation in the studied cell lines, and a good candidate to be targeted by chemotherapeutical approaches. TGF &#946; treatment showed discrepancies, revealing diverse behaviors in the different cell lines. An exhisting relation between TGF &#946; receptors and pAkt pathway may not be discharged, requiring further studies. The low occurrence of apoptosis reinforces this possibility, showing how important is Akt and related pathways for neoplastic progression, and its possible relation to cellular events not described to date.

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Das Mammakarzinom ist weltweit eine der häufigsten Malignomerkrankung der Frau. Im fortgeschrittenen Stadium der Erkrankung entwickeln bis zu 75 % der Patientinnen ossäre Metastasen. Basierend auf der Erkenntnis, dass GnRH-Analoga in vitro die Migration und Invasion von Mammakarzinomzellen während der Kokultur mit humanen Osteoblasten bzw. der Osteoblasten-ähnlichen Zellreihe MG-63 hemmen, sollten in dieser Arbeit die dem zugrundeliegenden molekularen Mechanismen näher betrachtet werden. Es ist bekannt, dass GnRH-Analoga in vielen verschiedenen GnRH-Rezeptor exprimierenden Tumorzelllinen die Wachstumsfaktor- und Wachstumsfaktorrezeptor-Expression beeinflussen und somit z.B. antiproliferative oder Apoptose-induzierende Effekte vermitteln. In dieser Arbeit wurde die humane Mammakarzinomzelllinie MCF-7 mit der osteoblasten-ähnlichen Zellreihe MG-63 kokultiviert. Zu bestimmten Zeitpunkten erfolgte die Behandlung der Mammakarzinomzellen mit drei verschiedenen GnRH-Analoga. Dem GnRH-I-Agonisten Triptorelin, dem GnRH-I-Antagonisten Cetrorelix und dem GnRH-II-Agonisten [D-Lysin]-GnRH-II. Neben einer nicht-kokultivierten Kontrolle jeder Zelllinie wurde eine nicht behandelte Kokulturkontrolle mitgeführt. Nach 48 h (bzw. nach 72 h od. 96 h) wurde der Versuch beendet und die mRNA-Expression von EGF, EGFR, TGF-beta und TGFBRI und -II in MCF-7 und MG-63 mittels PCR bestimmt. Zudem wurde die mRNA-Expression von EGF, EGFR und TGF-beta auch nach längerer Kokultur- und Behandlungszeit sowie unter Stressbedingungen beobachtet. Es hat sich gezeigt, dass es weder in MG-63 noch in MCF-7 signifikante Expressionsunterschiede von EGF, EGFR und TGF-beta im Vergleich zur Kokulturkontrolle gab. Dies hat sich auch weder im Zeitverlauf noch unter Stressbedingungen geändert. In MG-63 konnte man ebenfalls keine Expressionsunterschiede der TGFB-Rezeptoren beobachten. Dagegen konnte erstmals gezeigt werden, dass alle drei GnRH-Analoga die mRNA-Expression des TGFBR-I und -II während der Kokultur mit der osteoblasten-ähnlichen Zellreihe MG-63 signifikant hemmen. Während man in anderen Gewebetypen bereits eine verminderte Expression von TGFBR-I und -II nach Behandlung mit GnRH-Analoga nachweisen konnte, ist dies erstmals in einer Mammakarzinomzelllinie gelungen. Desweiteren kann man aus den vorliegenden Ergebnissen den Schluss ziehen, dass die verminderte Expression der TGFB-Rezeptoren eine Rolle in der bereits nachgewiesene GnRH-Analoga-vermittelten Migrations- und Invasionshemmung spielt.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Ovulation is controlled by a complex and dynamic interaction of factors, including endocrine and vasoactive mechanisms, cellular messengers and activating enzymes. It is well established that locally produced factors exert pivotal roles during ovulation. Some peptides such as angiotensin II (Ang II) and Natriuretic Peptides (NPs) have been prominent in local regulation of reproductive functions of mammals, beyond its systemic activities. Unlike angiotensin II (AngII), the role of angiotensin-(1-7) [Ang-(1-7)] has not been characterized in the ovary of mono-ovulatory species. In the first study, was evaluated the effect of Ang-(1-7) and its receptor (MAS) in the regulation of the ovulatory cascade. For this we used an in vitro model of supplementation with Ang- (1-7) or blocking its receptor on EREG mRNA expression in granulosa cells. Using an in vivo model, the intrafollicular injection of Ang-(1-7) antagonist (A-779 - MAS receptor inhibitor) was performed to evaluate the ovulation rate. Results showed that the Ang- (1-7) and A-779 inhibitor when in vitro culture, were not able to regulate to EREG mRNA expression. Likewise, the intrafollicular injection of A-779 did not block ovulation before the expected time of LH peak, suggesting that Ang-(1-7) has no role in the early ovulatory cascade in cattle. In the second experiment, the aim was to evaluate the pattern of mRNA expression in bovine granulosa cells for the Natriuretic Peptides Precursors (NPPs), receptors (NPRs) and key enzymes of this system after GnRH-induced ovulation in vivo. Using in vivo model main results have been shown the presence of several components of NPs system during ovulation in cattle, as well as increased mRNA expression for NPPC (natriuretic peptide precursor C) in granulosa cells after induction of ovulation with GnRH / LH. These results provide the first evidence that the C-type natriuretic peptide (NPC) is upregulated by LH and may be involved in ovulation and bovine luteinizing. Third study was conducted to answer like LH controls the mRNA expression for NPPC if this is up-regulated through EGF receptor (EGF-R). Moreover, we evaluated if the NPC regulates genes in ovulatory cascade and blockade EGF-R is capable of altering ovulation rate. For this we used an in vitro model of granulosa cell culture from large follicles (> 12 mm) and in vivo models to study ovulation, combined with intrafollicular injection and follicular dynamics and / or ovariectomy at strategic times. Our main results were: NPC does not regulate mRNA expression for amphiregulin (AREG) and EREG in vitro; NPPC mRNA coding is regulated by EGF-R but block EGF-R was not able to change the ovulation rate in bovines. These results suggest that the regulation and function of NPs during ovulation may differ between monovular and polyovular species. / A ovulação é controlada por uma complexa e dinâmica interação de fatores, incluindo mecanismos endócrinos e vasoativos, mensageiros celulares e enzimas ativadoras. Fatores produzidos localmente exercem papel essencial durante o período ovulatório. Alguns peptídeos como Angiotensina II (AngII) e os Peptídeos Natriuréticos (NPs) têm se destacado na regulação local das funções reprodutivas, além de suas atividades sistêmicas. Ao contrário da AngII, a função da Angiotensina-(1-7) [Ang-(1-7)] ainda não está caracterizada no ovário de espécies monovulatórias, como o bovino. No primeiro estudo foi avaliado o efeito da Ang-(1-7) e de seu receptor (MAS) na regulação da ovulação. Para isso foi realizada a suplementação com Ang-(1-7) ou bloqueio de seu receptor, em células da granulosa cultivadas in vitro, sobre a expressão de RNAm para epirregulina (EREG marcador inicial do processo de ovulação). Utilizando um modelo in vivo, foi realizada a injeção intrafolicular do inibidor do receptor MAS (A-779) para avaliar a taxa de ovulação. Os resultados demonstraram que a Ang-(1-7) e o inibidor A-779 quando em cultivo in vitro, não foram capazes de regular a expressão de RNAm para EREG. Da mesma forma que injeção intrafolicular de A-779 não inibiu o processo ovulatório, indicando que a Ang-(1-7) não possui papel relevante no início da cascata ovulatória em bovinos. No segundo estudo o objetivo foi caracterizar a expressão dos NPs, seus receptores e enzimas convertases durante a ovulação induzida a partir de GnRH/LH em bovinos. Utilizando modelo in vivo foram demonstrados como principais resultados a presença de vários componentes do sistema NPs durante a ovulação em bovinos, bem como um aumento na expressão de RNAm para NPPC (precursor do peptídeo natriurético tipo C) após indução da ovulação GnRH / LH em células da granulosa. Esses resultados fornecem a primeira evidência que o peptídeo natriurético tipo C (NPC) é regulado positivamente pelo LH e pode estar envolvido na ovulação e luteinização de bovinos. O terceiro estudo foi realizado para responder como ocorre a regulação do RNAm para NPPC pelo LH, se essa regulação é via receptor de EGF (EGF-R). Além disso, avaliamos se o NPC regula genes da cascata ovulatória e se o bloqueio EGF-R é capaz de alterar a taxa de ovulação. Para isso foram utilizados modelos in vitro de cultivo de células da granulosa de folículos grandes (> 12 mm) e modelos in vivo para estudo da ovulação aliados à injeção intrafolicular com ovariectomia em momentos estratégicos e/ou dinâmica folicular. Nossos principais resultados foram: NPC não regula a expressão de RNAm para anfirregulina (AREG) e EREG in vitro; RNAm para NPPC é regulado via EGF-R porém o bloqueio de EGF-R não foi capaz de alterar a taxa de ovulação em bovinos. Estes resultados sugerem que a regulação e função dos NPs durante a ovulação pode diferir entre espécies mono e multiovulatórias.

Durch Analyse der Subtraktion-cDNA Bibliothek einer humanen Lungentumor Zelllinie haben wir ein neues Mitglied der S100 Genfamilie identifiziert und charakterisiert, welches S100A14 benannt wurde. Die vollständige cDNA hat eine Länge von 1067 bp und kodiert für ein Protein von 104 Aminosäuren, welches die S100-spezifische Kalzium-bindende Domäne enthält. Das Gen wird in normalen humanen Epithelien ubiquitär exprimiert, zeigt jedoch Expressionsverluste in vielen Tumorzelllinien. Im Gegensatz zu Tumorzelllinien ist S100A14 auf mRNA- und Proteinebene in vielen humanen Primärtumoren stärker exprimiert, unter anderem in Lungen- und Brustkarzinomen. Um den Mechanismus der erhöhten S100A14 Expression in Lungen- und Brustkarzinomen zu verstehen, haben wir die Effekte des EGF (epidermal growth factor) und des TGF-alpha (transforming growth factor-alpha) untersucht. Beide Faktoren sind Liganden des ERBB Rezeptors und induzieren in der immortalisierten bronchialen Epithelzelllinie S100A14 Expression. Unter Verwendung spezifischer Inhibitoren konnte gezeigt werden, dass für die EGF-vermittelte transkriptionelle Induktion der ERK1/2 Signalweg (extracellular signal-regulated kinase) verantwortlich ist und eine de novo Proteinsynthese erfordert. Diese Ergebnisse unterstützend konnte immunhistologisch eine signifikante Korrelation zwischen der Überexpression von ERBB2 und S100A14 in primären Brustkarzinomen nachgewiesen werden. Phorbolester-12-Myristat-13-Acetat (PMA) verstärkte gleichfalls die S100A14 mRNA Expression in 9442 Zellen, was eine Regulation durch die Protein Kinase C (PKC) vermuten lässt. Die PMA-induzierte Expression von S100A14 wird ebenso wie die TGF-alpha/EGF-Induktion durch die Aktivierung des ERK1/2 Signalweges vermittelt. In Anbetracht der großen Bedeutung der ERK1/2 und PKC Signalwege in der Tumorentstehung und Tumorprogression ist zu vermuten, dass S100A14 über die aberrante Regulation dieser Signalwege an die maligne Transformation gekoppelt ist. / By analysing a human lung tumour cell line subtraction cDNA library, we have identified and characterized a novel member of the human S100 gene family that we designated S100A14. The full-length cDNA is 1067 bp and encodes a putative protein of 104 amino acids. The predicted protein contains the S100-specific EF-hand calcium-binding domain. The gene is ubiquitously expressed in normal human tissues of epithelial origin. S100A14 transcript was found to be down-regulated in many immortalized and tumour cell lines from diverse tissues. In contrast to the tumour cell lines, S100A14 shows up-regulation at the mRNA and protein level in many human primary tumours, including lung and breast carcinomas. To elucidate mechanisms whereby S100A14 expression is enhanced in lung and breast tumours, we studied the effects of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) on its expression. Both are ligands of ERBB receptor and induced S100A14 expression in the immortalized bronchial epithelial cells. By use of specific inhibitors, we found that EGF-mediated transcriptional induction of S100A14 involves extracellular signal-regulated kinase (ERK1/2) signalling and requires de novo protein synthesis. In support of these findings, we demonstrated by immunohistochemistry a significant correlation between ERBB2 and S100A14 protein overexpression in primary breast carcinomas. Our studies showed that the phorbol ester 12-myristate 13-acetate (PMA) increases S100A14 mRNA expression in immortalized bronchial epithelial cells suggesting regulation by protein kinase C (PKC). Similar to TGF-alpha/EGF induction, the PMA-induced S100A14 expression was also mediated by activation of the ERK1/2 signalling cascade. Considering the importance of the ERK1/2 and PKC signalling pathways in tumour development and progression we suggest that it is the aberrant regulation of these signalling cascades that couples S100A14 to malignant transformation.

Dans un premier temps, nous avons réalisé des revues systématiques de la littérature avec méta-analyses des données de survie. Ceci nous a conduits à sélectionner 4 marqueurs de mauvais pronostic pour la survie des CBNPC: le récepteur au facteur de croissance épidermique (EGF-R), un autre récepteur de cette famille (c-erbB-2) ainsi que deux autres facteurs potentiellement témoins de leur activité, Ki-67 (impliqué dans la prolifération) et le nombre des micro-vaisseaux (témoins de la néoangiogenèse). Dans une deuxième phase, nous avons étudié au laboratoire diverses questions sur des tumeurs bronchiques invasives. Premièrement, nous avons investigué le mécanisme de surexpression d’EGF-R et de c-erbB-2 et évalué si des anomalies génétiques pouvaient prédire cette surexpression, en recourant à des techniques d’immunohistochimie et de FISH. Ceci nous a permis d’observer que, si la majorité des CBNPC réséqués présentent des anomalies génétiques d’EGF-R et/ou de c-erbB-2, une amplification de ces gènes n’est présente que dans une minorité d’entre eux et n’est pas strictement corrélée à l’expression protéique. D’autre part, la survie de ces patients exprimant ou ayant une anomalie génique d’EGF-R et/ou c-erbB-2 est plus courte sans atteindre le seuil de signification statistique. Deuxièmement, nous avons recherché sur des tumeurs opérées d’éventuels liens entre les expressions d’EGF-R, de c-erbB-2 et de Ki-67. Aucune corrélation n’a été mise en évidence entre l’expression de ces 3 facteurs. Par contre, chez ces patients, l’expression de Ki-67 dans la tumeur s’est avérée être un facteur de mauvais pronostic pour la survie. Troisièmement, nous avons voulu savoir si un de ces marqueurs (EGF-R) présentait une valeur pronostique dans un groupe plus restreint de tumeurs plus avancées, les CBNPC de stade III. Pour mener cette recherche sur des biopsies, nous avons d’abord démontré que l’évaluation des marqueurs biologiques (EGF-R, c-erbB-2 et Ki-67) sur biopsie ne différait pas de celle réalisée sur des tumeurs réséquées. Comme les résultats étaient équivalents, nous avons pu étudier EGF-R sur les biopsies de CBNPC au stade III et montrer qu’EGF-R n’était pas un facteur pronostique pour la survie dans ce groupe assez homogène de tumeurs avancées. Dans la dernière phase, nous avons étudié des lésions représentatives des différents stades prénéoplasiques et néoplasiques précoces radiooccultes. Ces lésions ont été prélevées lors d’examens endoscopiques de photodétection. EGF-R, c-erbB-2, Ki-67 et le nombre des micro-vaisseaux ont été étudiés par immunohistochimie dans ces différents stades de lésions prénéoplasiques et néoplasiques précoces. Nous avons observé qu’EGF-R et Ki-67 sont statistiquement plus exprimés dans les dysplasies sévères et les carcinomes in que dans les dysplasies légères suggérant que, au moins pour ces 2 marqueurs, les dysplasies sévères se rapprochent plus des carcinomes in situ que des dysplasies légères. Alors que l’expression d’EGF-R est présente dès le stade de dysplasie sévère, une augmentation du nombre des micro-vaisseaux n’est présente qu’au stade de tumeurs micro-invasives. C-erbB-2 n’est quant à lui pas exprimé dans ces lésions bronchiques prénéoplasiques et néoplasiques précoces. En conclusion, les facteurs biologiques, EGF-R, c-erbB-2 et Ki-67 et le nombre des micro-vaisseaux s’avèrent des facteurs de mauvais pronostic dans le CBNPC. La surexpression d’EGF-R et de c-erbB-2 dans les cancers réséqués résulte très rarement d’une amplification génique et nous n’avons pas trouvé dans ces tumeurs de corrélation entre l’expression des marqueurs moléculaires étudiés. Dans les tumeurs plus avancées de stade III, EGF-R n’est pas un facteur discriminant pour le pronostic. Les anomalies de certains de ces marqueurs (EGF-R et Ki-67) apparaissent précocement, dès les stades prénéoplasiques, avec un seuil se situant entre les lésions bronchiques de bas et de haut grades. La néoangiogénèse, évaluée par le nombre des micro-vaisseaux, s’observe à partir des cancers micro-invasifs tandis que c-erbB-2 n’apparaît qu’au stade invasif. Dans la séquence d’apparition des anomalies génétiques conduisant au cancer invasif, l’atteinte d’EGF-R précède la néoangiogénèse.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common type of cancer in the western hemisphere with a five-year survival rate of only 50% for patients with a localized tumor, which decreases significantly to as low as 5% for those patients with tumors that have metastasized to distant sites of the body. It has been found that both mutant p53 and epidermal growth factor receptor (EGFR) signaling pathways function to increase the expression of CXCL5, which has been identified as a key mediator in the process of tumor metastasis. Previous data from our lab suggested that the p53 homolog, p63, may function as a negative regulator of CXCL5 and that mutant p53 may inhibit this molecule to elevate CXCL5 expression levels. In the current study we utilized an model system in which the H179L p53 mutant was expressed in HN4 cells to investigate the hypothesis that mutant p53 enhances expression of CXCL5 by both interfering with p63 function and cooperating with EGFR/EPS8 signaling, leading to increased cell proliferation and motility. The results of the current study indicate a role for mutant p53 in head and neck squamous cell carcinoma proliferation, migration and tumorigenicity, possibly through enhancement of CXCL5 expression. We were able to show that mutant p53 expression caused an increase in the expression of this chemokine in addition to increasing proliferation and migration of the cells compared to the vector control. Additionally, we showed that p63 protein is a negative regulator of CXCL5 that is downregulated in the cells expressing mutant p53, which suggests that through direct interaction, mutant p53 may function to inhibit p63 function as well as target it for degradation. These results support the hypothesis that GOF mutant p53 enhances expression of CXCL5 by interfering with p63 function in cancer cells. The results of the current study results also showed that upon treatment with EGF, HN4 cells expressing mutant p53 express elevated levels of CXCL5; and that the mutant p53-expressing HN4 cells cooperate with EGFR/EPS8 signaling to further deregulate chemokine expression. These data taken together suggest there are complex interactions taking place between mutant p53, p63, EGFR signaling, and CXCL5 to regulate the biological processes that promote tumor progression that could lead to metastasis. Additional studies are needed to further elucidate the molecules involved in the mutant p53 mechanism that promotes tumorigenesis.

Les patients atteints de cancers reçoivent différents traitement, tels que la radiothérapie ou la chimiothérapie. Actuellement, environ 60% des enfants survivants du cancer développent des effets secondaires cognitifs, consécutifs aux traitements énoncés précédemment. Compte tenu de la perspective du développement psychomoteur de l’enfant et de l’immaturité du système nerveux central (SNC) chez ces patients, il s’avère particulièrement pertinent d’étudier les effets secondaires que provoquent les traitements anticancéreux sur le développement cognitif de cette population de malades. Des études ont démontrées l’existence de liens étroits entre ces effets secondaires et l’abolition de la neurogénèse provoquée principalement par l’irradiation. Ce projet de maîtrise porte sur les effets du facteur de croissance épidermique, l’EGF (un facteur de croissance impliqué dans la prolifération cellulaire) sur la neurogénèse de la souris. Nous avons également cherché un vecteur de sécrétion efficace pour permettre une diffusion continue d’EGF à long terme (2 à 4 semaines). Notre hypothèse est que l’EGF serait capable de stimuler la neurogénèse et protéger les cellules de l’apoptose dans le cerveau de la souris, suite à une irradiation. Nous avons montré un effet positif de l’EGF sur la formation et la prolifération des neuroblastes Dcx(+) dans la zone sous ventriculaire (ZSV) et non dans l’hippocampe (Hi), suite à l’injection de l’EGF, directement dans le cerveau à l’aide d’une pompe osmotique. Nous avons observé que cette augmentation de la quantité de jeunes neurones est indépendante de la capacité de l’EGF à les protéger de l’apoptose. L’EGF ne protège pas non plus les blastes leucémiques, issus de lignées de cellules humaines, des effets secondaires d’une irradiation. Les cellules souches mésenchymateuses (CSM) modifiées génétiquement et générées pour sécréter l’EGF ne montrent aucun effet sur la stimulation de la neurogénèse quand elles sont directement injectées dans le cerveau. Finalement, nos résultats indiquent que l’EGF pourrait être un bon candidat pour le développement de nouvelles thérapies pour traiter les effets secondaires que provoque une irradiation du cerveau. L’utilisation de pompes pour permettre l’administration d’EGF dans le cerveau devient alors très intéressante pour améliorer la qualité de vie des patients. / Patients with cancer receive different treatments such as radiotherapy (ionizing radiation) or chemotherapy. Currently about 60% of children surviving cancer are prone to the development of treatment-related delayed side effects, such as neurocognitive impairments. Given the prospect of psychomotor development of children and central nervous system (CNS) immaturity in these patients, it is especially relevant to study the adverse effects related to cognitive impairment caused by cancer treatments on this population of patients. Studies have shown the existence of close links between this delayed side effects and the abolition of neurogenesis caused mainly by irradiation. This masters degree project concerns the effects of epidermal growth factor, EGF (a growth factor involved in cell proliferation), on neurogenesis in the C57BL/6 mice, which drastically decrease after exposure to ionizing radiation. We also sought a vector of efficient secretion to allow continuous long-term secretion of EGF (2-4 weeks). Our hypothesis is that EGF will stimulate neurogenesis and protect cells from apoptosis in the brain of mice following radiation. We have shown a positive effect of EGF on the formation and proliferation of young neurons Dcx (+) in the subventricular zone (SVZ) but not in the hippocampus (Hi) following EGF injection directly in the brain using an osmotic pump. We also observed that the increase of young neurons is independent of the ability of EGF to protect them from apoptosis. Moreover, EGF doesn’t protect leukemic human blasts after irradiation. Mesenchymal stem cells (MSC) genetically engineered to secrete EGF did not stimulate neurogenesis when injected directly in the brain. Finally our results indicate that EGF could be a good candidate for the development of new therapies to treat the side effects that irradiation causes in the brain. The use of pumps to allow the administration of recombinant EGF in specific brain regions becomes very interesting. Indeed this delivery system is increasingly used and effective to help improve the quality of life of patients.